The present invention relates to a novel nonhemolytic microorganism and to its utilization, and more particularly to a novel microorganism which expresses not only high antimicrobial activity against those noxious bacteria which give off offensive odors in the course of proliferation (for example, Staphylococcus aureus, Klebsiella neumoniae, etc.) and, as such, is capable of getting rid of the malodor originating from effluent treatment and facilities, but also high antimicrobial activity against other toxic bacteria (for example, meticillin-resistant Staphylococcus aureus, pathogenic Escherichia coli O-157, Legionella pneumophila, etc.), thus finding application in a broad spectrum of uses and to the utilization thereof.
It should be understood that the antimicrobial activity of the novel microorganism according to the present invention is derived not only from the extracellular and intracellular secretions of this novel microorganism but also from the following mechanism.
The novel microorganism of the present invention proliferates at an unusually high growth rate. Thus, at an incubation temperature of 20xcx9c40xc2x0 C., it multiplies from an initial population of 103 cells/g (ml) to a population 106 cells/g (ml) in about 2 hours and further to 108 cells/g (ml) within 6 hours. Whereas growth of ordinary or adventitious bacteria occurs only after the so-called induction period, that is to say an acclimatization period (the time which a microorganism placed in a new environment requires for its being acclimatized to the new environment) of about 6 hours, the novel microorganism of the present invention has substantially no xe2x80x9cinduction periodxe2x80x9d as mentioned above but undergoes cell division and multiplies rapidly in an explosive manner after initiation of culture. As the microorganism of the present invention avariciously digests and assimilates available nutrients and grows explosively while said other adventitious bacteria are still in the induction period of growth, proliferation of the adventitious bacteria is inhibited and the above-mentioned antimicrobial activity against such adventitious bacteria is expressed. Thus, this novel microorganism having substantially no induction period and starting to grow immediately after commencement of culture inhibits growth of noxious bacteria.
Recent years have witnessed what may be called the heyday of the technology utilizing microorganisms, inclusive of the waste water treatment technology. While a large variety of microorganisms are utilized in waste water treatment, the following can be reckoned as representative organisms. Thus, as the bacteria used, Zooglea, Sphaerotilus, etc. can be mentioned; as the protozoa used, Rhizopoda, Mastigophora, Ciliata, etc. can be mentioned; and as the algae, Cyanophyceae, Chlorophyceae, Diatomeae, etc. can be mentioned.
These microorganisms are capable of decomposing organic matter and some of inorganic matter but tend to induce, in decomposing them, the production of malodors (ammonia odor, hydrogen sulfide odor) and malodor-emitting substances, and the malodors generated from waste water treatment plants have frequently been reported as a social problem. Thus, even if the decomposition of organic and inorganic substances were efficiently achieved, the malodors originating from such treatment facilities remained to be dealt with. Particularly, the active sludge formed in a sedimentation tank becomes progressively anaerobic and, as anaerobic bacteria grow, they emit a copious malodor. In the course of recycling such an active sludge to the aeration tank, a considerable spread of noxious odor drifts around the effluent treatment plant.
With the genesis of the foregoing problem by way of background, the inventors of the present invention explored earnestly for a new useful microorganism and ultimately succeeded in isolating from soil a novel useful microorganism which does not cause evolution of the malodors of hydrogen sulfide, methylmercaptan, etc. in the decomposition of organic matter and has antimicrobial activity not only against the bacteria which grow with emission of malodors, such as Staphylococcus aureus and Klebsiella pneumoniae, but also against meticillin-resistant S. aureus, pathogenic E. coli. O-157, Legionella spp., Pseudomonas aeruginosa and so forth. They found, also, that this novel microorganism can be utilized in a large variety of uses and have ultimately developed the present invention.
The novel microorganism of the present invention is a nonhemolytic Bacillus subtilis-related microorganism which is not less than 4 times as large as the type culture strain of Bacillus subtilis (IFO 3134) in cell volume and grows substantially without an induction period in an early stage of culture, for example showing an increase in population from an initial count of 103 cells/ml to a count of not less than 107 cells/ml in 4 hours when it is subjected to nutrient broth shake culture at 37xc2x0 C. As the synergism of those characteristics, the metabolic energy generated by its growth reaches not less than 1xc3x97104 times as compared with said type culture strain of B. subtilis. As such, this microorganism expresses antimicrobial activity against Staphylococcus aureus, Klebsiella pneumoniae, meticillin-resistant S. aureus, pathogenic E. coli O-157, Legionella spp., Pseudomonas aeruginosa, Fusarium spp., koji mold (Aspergillus oryzae), blue mold (Penicillium spp.), Trichophyton spp., and Rhyzopus spp.
As examples of the Bacillus subtilis-related microorganism, which is the novel microorganism of the present invention, the following OYK strains (deposited under the international convention with Patent Microorganism Deposit Center, National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, the Ministry of International Trade and Industry, Japan) can be mentioned.
(1) Bacillus sp. OYK-01-600 (FERM BP-6394),
(2) Bacillus sp. OYK-03-600 (FERM BP-6395),
(3) Bacillus sp. OYK-04-000 (FERM BP-6396).
In the following description, each of OYK-01-600, OYK-03-600 and OYK-04-000 will be referred to briefly as the OYK strain.
The OYK strain digests protein and grows luxuriantly. Moreover, the OYK strain is characterized in that it does not produce hemolysin and, therefore, is highly safe to the human being. The safety of the strain has been confirmed by administering it to animals by four routes, namely intravenous, airway, oral and percutaneous.
The OYK strain is comparatively large in size among various strains of B. subtilis. For example, it is about 4 times (by volume) larger than the type culture strain of Bacillus subtilis [IFO 3134 (0.4xcx9c0.6xc3x971.5xcx9c3.2 xcexcm)]. Moreover, the OYK strain has a greater initial growth potential after the start of culture and thereby produces higher metabolic energies (whereas the type culture strain multiplies only 6-fold in 4 hours after the start of culture, the OYK strain grows as many as about 80,000 times). Moreover, being a facultative anaerobe, the strain is able to grow actively under both aerobic and anaerobic conditions. Therefore, it increases the temperature of organic wastes quickly in the production of a compost to promote growth of the thermophilic actinomyces and hyphomyces and thereby reduce the compost ripening time and give rise to a full-ripe compost.